The present invention pertains to endocrinology, medicine, and cell biology. More specifically, this invention relates to the endogenous stimulation of production of insulin-like growth factor I (“IGF-I”) in a subject at a level greater than non-treated subjects. Administration of DNA encoding IGF-I helps enhance angiogenesis and myogenesis. It also upregulates angiogenic factors, upregulates angiopoietins, and treats complications of diabetes.
IGF-I has important growth promoting and metabolic effects and is expressed in virtually every tissue of the body. The highest expression is found in the liver. The effects of liver-derived and systemically secreted IGF-I are predominantly endocrine, while locally produced IGF-I in peripheral tissues has more of an autocrine or paracrine effect. Recent studies have been aimed to elucidate the role and effects of systemically or locally produced IGF-I. Data in the literature suggests that the liver-derived IGF-I is important for carbohydrate- and lipid-metabolism and for the regulation of GH-secretion at the pituitary level. Furthermore, it regulates adult axial skeletal growth and cortical radial growth while it is not required for appendicular skeletal growth, which is linked to locally-derived IGF-I (Sjogren et al., 1999; Sjogren et al., 2002).
The GHRH-GH-IGF-I production pathway is composed of a series of interdependent genes whose products are required for normal growth, development, regeneration and repair (Caroni et al., 1994). The pathway genes include: (1) ligands, such as growth hormone (“GH”) and IGF-I; (2) transcription factors such as prophet of Pit-1, or prop 1, and Pit-1; (3) agonists and antagonists, such as growth hormone releasing hormone (“GHRH”) and somatostatin (“SS”), respectively; and (4) receptors, such as the GHRH receptor (“GHRH-R”) and the GH receptor (“GH-R”). These genes are expressed in different organs and tissues, including the hypothalamus, pituitary, liver, and bone.
IGF-I is a 70 amino acid polypeptide with extensive structural homology to insulin (49%) and IGF-II (61%). Hormonally, IGF-I is regulated as part of the GHRH/GH axis. GHRH, secreted by the hypothalamus, stimulates release of GH by the anterior pituitary. GH subsequently stimulates production of IGF-I in the liver and other tissues. IGF-I provides negative feedback in this axis by directly inhibiting GH release from the pituitary and indirectly by inducing somatostatin (“SS”) expression by the hypothalamus (Vance, 1990). The possibility of a direct regulatory role of this axis with respect to any autocrine or endocrine effects of IGF-I remains to be proven. IGFs were originally thought to be liver-derived mediators of GH action, but now it is known that they are synthesized and secreted from many cell types, including muscle or bone (Florini et al., 1991). This factor mediates many of the growth-promoting effects of GH in postnatal animals by binding to the type I IGF receptor (“IGF-R”). In animal studies, administration of GH to hypophysectomized rats resulted in a significant increase in IGF-I mRNA in skeletal muscle. Implantation of GH secreting cells in non-growing rats caused a seven-fold increase in IGF-I mRNA and a 50% increase in the mass of the gastrocnemius muscles (Kelly et al., 1990). Increased expression of IGF-I genes by passive mechanical stretch or acute exercise shows a correspondence between muscle hypertrophy and IGFs (Vandenburgh et al., 1991). Targeted over-expression of IGF-I in skeletal muscle in transgenic animals enhances muscle growth (Coleman et al., 1995; Goldspink, 1999). Thus, IGF-I seems to be important in the hormonal regulation of skeletal muscle growth.
IGF-I provides an attractive candidate for therapeutic approaches on muscle and heart. IGF-I was shown to play an important role in the growth and regeneration of peripheral nerves and skeletal muscle, and it was investigated as a treatment for neuromuscular disorders (Cheng et al., 1996; Florini et al., 1993). In addition, expression of IGF-I in skeletal muscle is increased coincident with stretch-induced myofiber overloading and hypertrophy (Goldspink, 1999) and muscle regeneration following injury (Kasemkijwattana et al., 1998; Menetrey et al., 2000). Treatment with exogenous IGF-I protein reduces muscle degeneration and atrophy in dystrophic mice (De Luca et al., 1999; Hsu et al., 1997).
In addition to its role in myogenesis and nerve regeneration, IGF-I is also a potential angiogenic factor. Angiogenesis, the formation of neo-vessels from the endothelium of pre-existing vessels, plays an essential role in embryonic development and tissue repair (Folkman, 1995). Neo-vessels form in response to stimulation by soluble angiogenic factors, which regulate endothelial migration, proliferation, survival, and proteolytic activity (Folkman and Klagsbrun, 1987). The most well-studied factors described to date, vascular endothelial growth factor (“VEGF”), fibroblast growth factor (“bFGF”) and the angiopoietins (ANG-1, ANG-2), have emerged as critical regulators of the angiogenic process (Davis et al., 2003; Horvath et al., 2002). These molecules promote neo-vessel formation and morphogenesis by cooperating closely through a carefully orchestrated sequence of angioregulatory events (Peters, 1998; Veikkola et al., 2000). Current therapeutic angiogenesis strategy by using angiogenic growth factors had some success in treating ischemic disease, as peripheral diabetic disease, diabetic retinopathy or age-related macular degeneration (major causes of blindness in the western world), accelerate healing, as well as cardiac ischemic disease (Silvestre and Levy, 2002). As many as 10 million people in the USA have peripheral arterial disease (“PAD”) with more than 10% prevalence in people over 60 years old. Generally, men have a higher prevalence of PAD than women. The risk factors for PAD are similar to those for coronary artery disease (“CAD”) and cerebrovascular disease (“CBVD”), but diabetes and cigarette smoking have a particularly strong association with PAD (Beckman et al., 2002; Criqui 2001; Fowler et al., 2002).
IGF-I may be an initiator of the angiogenic process. IGF-I receptors have been shown to be present on endothelial cells of bone (Fiorelli et al., 1994; Fiorelli et al., 1996), retina (Spoerri et al., 1998), and aorta (Kobayashi and Kamata, 2002). IGF-I has also been shown to induce the expression of VEGF mRNA on retinal pigment epithelial cells (Punglia et al., 1997), osteoblasts (Akeno et al., 2002; Goad et al., 1996), vascular endothelial cells (Miele et al., 2000), and in a variety of tumor cells (Bermont et al., 2000; Reinmuth et al., 2002; Wu et al., 2002). IGF-I induces cell migration and tubular formation of cultured bovine retinal endothelial cells and human endothelial cells in vitro (Castellon et al., 2002; Shigematsu et al., 1999). Increased branching of aged cultured micro-vessels is enhanced by IGF-I (Arthur et al., 1998). IGF-I acts as a vasoactive factor by inhibiting vessel contraction, via stimulation of nitric oxide production (Walsh et al., 1996). During muscle regeneration, angiogenesis is induced in order to vascularize the growing muscle. IGF-I induces muscle satellite cell proliferation and differentiation in vivo (Rabinovsky et al., 2003) and it is known that IGF-I induces VEGF expresssion in satellite cells. Therefore, IGF-I initiates the angiogenic pathways that occur in injured muscle.
In additon to inducing aniogenesis in skeletal muscle, studies suggest that IGF-I may also induce aniogenesis in the heart. Studies show that IGF-I is capable of inducing a hypertrophic response in the heart by stimulating cardiac myocytes and fibroblasts to initiate a variety of processes associated with hypertrophy. IGF-I's activities were demonstrated in both in vitro and in vivo model systems. In cultured neonatal ventricular myocytes, the addition of IGF-I induces DNA synthesis (Kajstura et al., 1994; Kardami, 1990), the transcription of several genes associated with hypertrophy and hyperplasia, including myosin light chain-2, troponin and α-skeletal actin (Ito et al., 1993), and in vitro increased myofibril production (Donath et al., 1994; Donath et al., 1997). In vivo, IGF-I and its receptor are upregulated in cardiomyocytes of experimentally infarcted ventricles. This may be followed by DNA replication and mitotic division of a portion of the remaining cardiomyocytes (Reiss et al., 1994). IGF-I protects against apoptosis in cultured and primary cardiomyocytes (Wang et al., 1998b; Wang et al., 1998a) and in a mouse model of ischemic injury (Buerke et al., 1995). A transgenic mouse with increased IGF-I serum levels exhibits cardiomyocyte hyperplasia but no hypertrophy (Reiss et al., 1996). IGF-I and GH have been shown to improve cardiac performance in both experimental cardiac failure (Duerr et al., 1996) and that developed in human patients (Donath et al., 1998). Consequently, IGF-I and GH are seriously considered as potential therapeutic agents for situations in which hypertrophy and/or hyperplasia of cardiomyocytes would be desirable, such as following myocardial infarction or in hypocontracting cardiomyopathies (Lombardi et al., 1997). Adequate vascularization of the myocardium in this case is critical.
Although previous research demonstrated that IGF-I has potential for treatment of different conditions, systemic administration of IGF-I protein may require frequent dosing and elicit numerous side effects. For instance, recombinant IGF-I given to diabetic patients resulted in adverse effects such as edema and tachycardia (Jabri et al., 1994). Increased serum IGF-I levels may also accelerate the progression of diabetic nephropathy (Zhuang et al., 1996) and proliferative retinopathy (Glazner et al., 1994). In contrast to systemic delivery of IGF-I protein, non-viral IGF-I gene delivery targeted to skeletal muscle offers the potential to provide sustained and localized expression of IGF-I with infrequent administration and with minimal systemic side effects (Alila et al., 1997).
During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. The injection of a recombinant adeno-associated virus directing over-expression of IGF-I in differentiated muscle fibers promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and prevents aging-related muscle changes in elderly adult mice, resulting in a 27% increase in strength as compared with uninjected aged muscles. Muscle mass and fiber-type distributions are maintained at levels similar to those in young adults. These effects may primarily be due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I (Barton-Davis et al., 1998).
Muscle injuries are a challenging problem in traumatology, and the most frequent occurrence in sports medicine. In mice, massive muscle regeneration occurs in the first 2 weeks post injury that is subsequently followed by the development of muscle fibrosis. Growth factors, as bFGF, IGF-I, and NGF are capable of stimulating myoblast proliferation and differentiation in vitro and improving the healing of the injured muscle in vivo. Adenoviruses have been used to mediate direct and ex vivo gene transfer of these growth factors in the injured muscle (Kasemkijwattana et al., 1998). Liposome IGF-I gene transfer accelerates wound healing in burned rats and attenuates deleterious side effects associated with high levels of IGF-I. Rats receiving weekly subcutaneous injections of liposomes and IGF-I constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (Jeschke et al., 1999). Intramuscular injection of a plasmid encoding human IGF-I (“hIGF-I”) and engineered to restrict expression to skeletal muscle produced sustained local concentrations of biologically active hIGF-I. When normal rats received a single intramuscular injection of plasmids formulated as a complex with polyvinylpyrrolidone (“PVP”), the results showed that hIGF-I mRNA and hIGF-I protein were detectable in the injected muscles for the duration of the study. Biological activity of hIGF-I was determined by immunodetection of a nerve-specific growth-associated protein, GAP43, an indicator of motor neuron sprouting (Alila et al., 1997).
Gene transfer into skeletal muscle holds promise for the treatment of a variety of serum protein deficiencies, muscular dystrophies, and chronic ischemic limb syndromes. It is currently being developed as a method for the production, secretion and delivery of physiologically active proteins as hormones and may ultimately be applied to the treatment of several diseases (MacColl et al., 1999). The past few years have seen the development of new and improved vectors for programming recombinant gene expression in skeletal muscle. Important advances include first, novel plasmid DNA, adenovirus, and adeno-associated virus vectors that can be used to express stably therapeutic levels of recombinant proteins in the skeletal muscle of immunocompetent hosts and second, the development of vector systems that enable regulated and tissue-specific transgene expression in skeletal muscle in vivo.
Direct plasmid DNA gene transfer is currently the basis of many emerging nucleic acid therapy strategies and thus does not require viral genes or lipid particles (Aihara and Miyazaki, 1998; Muramatsu et al., 2001). Skeletal muscle is the target tissue of choice, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that express over months or years in an immunocompetent host (Davis et al., 1993; Tripathy et al., 1996).
Recently, the delivery of specific genes to somatic tissue in a manner that can correct inborn or acquired deficiencies and imbalances was proven to be possible (Herzog et al., 2001; Song et al., 2001; Vilquin et al., 2001). Gene-based drug delivery offers a number of advantages over the administration of recombinant proteins. These advantages include the conservation of native protein structure, improved biological activity, avoidance of systemic toxicities, and avoidance of infectious and toxic impurities. In addition, nucleic acid vector therapy allows for prolonged exposure to the protein in the therapeutic range, because the newly secreted protein is present continuously in the blood circulation. In a few cases, the relatively low expression levels achieved after simple plasmid injection are sufficient to reach physiologically acceptable levels of bioactivity of secreted peptides, especially for vaccine purposes (Danko and Wolff, 1994; Tsurumi et al., 1996).
The primary limitation of using recombinant protein is the limited availability of protein after each administration. Nucleic acid vector therapy using injectable DNA plasmid vectors overcomes this, because a single injection into the patient's skeletal muscle permits physiologic expression for extensive periods of time (WO 99/05300 and WO 01/06988). Injection of the vectors promotes the production of enzymes and hormones in animals in a manner that more closely mimics the natural process.
In a plasmid-based expression system, a non-viral gene vector may be composed of a synthetic gene delivery system in addition to the nucleic acid encoding a therapeutic gene product. In this way, the risks associated with the use of most viral vectors can be avoided, including the expression of viral proteins that can induce immune responses against target tissues and the possibility of DNA mutations or activations of oncogenes. The non-viral expression vector products generally have low toxicity due to the use of “species-specific” components for gene delivery, which minimizes the risks of immunogenicity generally associated with viral vectors. Additionally, no integration of plasmid sequences into host chromosomes has been reported in vivo to date, so that this type of nucleic acid vector therapy should neither activate oncogenes nor inactivate tumor suppressor genes. As episomal systems residing outside the chromosomes, plasmids have defined pharmacokinetics and elimination profiles, leading to a finite duration of gene expression in target tissues.
Among the non-viral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. However, the use of directly injectable DNA plasmid vectors has been limited in the past. The inefficient DNA uptake into muscle fibers after simple direct injection has led to relatively low expression levels (Prentice et al., 1994; Wells et al., 1997). In addition, the duration of the transgene expression has been short (Wolff et al., 1990). The most successful previous clinical applications have been confined to vaccines (Danko and Wolff, 1994; Tsurumi et al., 1996). A vector system for the delivery and controlled expression of recombinant IGF-I genes was previously described in U.S. patent application Ser. No. 09/861,101. This vector system included a 5′ flanking region with a naturally-occurring promoter, a linker region providing a site for insertion of a nucleic acid sequence and connecting the 5′ flanking region to the nucleic acid sequence, a nucleic acid sequence encoding IGF-I, and a 3′ flanking region. Administration of the vector system involved direct or intravenous injection and was shown to improve nerve regeneration, treat muscle atrophy, treat diabetes, treat osteoporosis, and improve livestock.
Efforts have been made to enhance the delivery of plasmid DNA to cells by physical means including electroporation, sonoporation, and pressure. Administration by electroporation involves the application of a pulsed electric field to create transient pores in the cellular membrane without causing permanent damage to the cell. It thereby allows for the introduction of exogenous molecules (Smith and Nordstrom, 2000). By adjusting the electrical pulse generated by an electroporetic system, nucleic acid molecules can travel through passageways or pores in the cell that are created during the procedure. U.S. Pat. No. 5,704,908 describes an electroporation apparatus for delivering molecules to cells at a selected location within a cavity in the body of a patient. These pulse voltage injection devices are also described in U.S. Pat. Nos. 5,439,440 and 5,702,384, and PCT WO 96/12520, 96/12006, 95/19805, and 97/07826.
Recently, significant progress to enhance plasmid delivery in vivo and subsequently to achieve physiological levels of a secreted protein was obtained using the electroporation technique. Electroporation has been used very successfully to transfect tumor cells after injection of plasmid (Lucas et al., 2002; Matsubara et al., 2001) or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors in humans (Gehl et al., 1998; Heller et al., 1996). Electroporation also has been extensively used in mice (Lesbordes et al., 2002; Lucas et al., 2001; Vilquin et al., 2001), rats (Terada et al., 2001; Yasui et al., 2001), and dogs (Fewell et al., 2001) to deliver therapeutic genes that encode for a variety of hormones, cytokines or enzymes. Previous studies using growth hormone releasing hormone (GHRH) showed that plasmid therapy with electroporation is scalable and represents a promising approach to induce production and regulated secretion of proteins in large animals and humans (Draghia-Akli et al., 1999; Draghia-Akli et al., 2002). Electroporation also has been extensively used in rodents and other small animals (Bettan et al., 2000; Yin and Tang, 2001). It has been observed that the electrode configuration affects the electric field distribution, and subsequent results (Gehl et al., 1999; Miklavcic et al., 1998). Preliminary experiments indicated that for a large animal model, needle electrodes give consistently better reproducible results than external caliper electrodes.
The ability of electroporation to enhance plasmid uptake into the skeletal muscle has been well documented, as described above. In addition, plasmid formulated with poly-L-glutamate (“PLG”) or polyvinylpyrrolidone (“PVP”) has been observed to increase plasmid transfection and consequently expression of the desired transgene. The anionic polymer sodium PLG could enhance plasmid uptake at low plasmid concentrations, while reducing any possible tissue damage caused by the procedure. PLG is a stable compound and resistant to relatively high temperatures (Dolnik et al., 1993). PLG has been previously used to increase stability in vaccine preparations (Matsuo et al., 1994) without increasing their immunogenicity. It also has been used as an anti-toxin after antigen inhalation or exposure to ozone (Fryer and Jacoby, 1993). In addition, plasmid formulated with PLG or PVP has been shown to increase gene transfection and consequently gene expression to up to 10 fold in the skeletal muscle of mice, rats and dogs (Fewell et al., 2001; Mumper et al., 1998). PLG has been used to increase stability of anti-cancer drugs (Li et al., 2000) and as “glue” to close wounds or to prevent bleeding from tissues during wound and tissue repair (Otani et al., 1996; Otani et al., 1998).
Although not wanting to be bound by theory, PLG increases the transfection of the plasmid during the electroporation process, not only by stabilizing the plasmid DNA, and facilitating the intracellular transport through the membrane pores, but also through an active mechanism. For example, positively charged surface proteins on the cells could complex the negatively charged PLG linked to plasmid DNA through protein-protein interactions. When an electric field is applied, the surface proteins reverse direction and actively internalize the DNA molecules, a process that substantially increases the transfection efficiency. Furthermore, PLG will prevent the muscle damage associated with in vivo plasmid delivery (Draghia-Akli et al., 2002a) and will increase plasmid stability in vitro prior to injection.
Although there are references in the art directed to electroporation of eukaryotic cells with linear DNA (McNally et al., 1988; Neumann et al., 1982; Toneguzzo et al., 1988; Aratani et al., 1992; Nairn et al., 1993; Xie and Tsong, 1993; Yorifuji and Mikawa, 1990), these examples illustrate transfection into cell suspensions, cell cultures, and the like, and the transfected cells are not present in a somatic tissue.
U.S. Pat. No. 4,956,288 is directed to methods for preparing recombinant host cells containing high copy number of a foreign DNA by electroporating a population of cells in the presence of the foreign DNA, culturing the cells, and killing the cells having a low copy number of the foreign DNA.
In summary, increased angiogenesis and myogenesis in a treated subject were previously restricted in scope. The related art has shown that it is possible to improve these different conditions in a limited capacity utilizing recombinant protein technology, but these treatments have some significant drawbacks. It has also been shown that nucleic acid expression constructs that encode recombinant proteins are viable solutions to the problems of frequent injections and high cost of traditional recombinant therapy. Unfortunately, each plasmid construct for a given recombinant protein must be evaluated individually, because the related art does not teach one skilled in the art to accurately predict how changes in structure (e.g. amino-acid sequences) will lead to changed functions (e.g. increased or decreased stability) of a recombinant protein. Therefore, the beneficial effects of nucleic acid expression constructs that encode expressed proteins can only be ascertained through direct experimentation. There is a need in the art for expanded treatments for subjects with a disease by utilizing nucleic acid expression constructs that are delivered into a subject and express stable therapeutic proteins in vivo.